Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD^+ve mult-HCL, IgD mediated persistent Ca²⁺ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD^−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.     

Design,synthesis, and evaluation of l-cystine diamides as l-cystine crystallization inhibitors for cystinuria

To overcome the chemical and metabolic stability issues of l-cystine dimethyl ester (CDME) and l-cystine methyl ester (CME), a series of l-cystine diamides with or without N^α-methylation was designed, synthesized, and evaluated for their inhibitory activity of l-cystine crystallization. l-Cystine diamides 2a–i without N^α-methylation were found to be potent inhibitors of l-cystine crystallization while N^α-methylation of l-cystine diamides resulted in derivatives 3b–i devoid of any inhibitory activity of l-cystine crystallization. Computational modeling indicates that N^α-methylation leads to significant decrease in binding of the l-cystine diamides to l-cystine crystal surface. Among the l-cystine diamides 2a–i, l-cystine bismorpholide (CDMOR, LH707, 2g) and l-cystine bis(N′-methylpiperazide) (CDNMP, LH708, 2h) are the most potent inhibitors of l-cystine crystallization.     

Minerals solubilizing and mobilizing microbiomes: A sustainable approach for managing minerals’ deficiency in agricultural soil

Agriculture faces challenges to fulfil the rising food demand due to shortage of arable land and various environmental stressors. Traditional farming technologies help in fulfilling food demand but they are harmful to humans and environmental sustainability. The food production along with agro-environmental sustainability could be achieved by encouraging farmers to use agro-environmental sustainable products such as biofertilizers and biopesticides consisting of live microbes or plant extract instead of chemical-based inputs. The eco-friendly formulations play a significant role in plant growth promotion, crop yield and repairing degraded soil texture and fertility sustainably. Mineral solubilizing microbes that provide vital nutrients like phosphorus, potassium, zinc and selenium are essential for plant growth and development and could be developed as biofertilizers. These microbes could be plant associated (rhizospheric, endophytic and phyllospheric) or inhabit the bulk soil and diverse extreme habitats. Mineral solubilizing microbes from soil, extreme environments, surface and internal parts of the plant belong to diverse phyla such as Ascomycota, Actinobacteria, Basidiomycota, Bacteroidetes, Chlorobi, Cyanobacteria, Chlorophyta, Euryarchaeota, Firmicutes, Gemmatimonadetes, Mucoromycota, Proteobacteria and Tenericutes. Mineral solubilizing microbes (MSMs) directly or indirectly stimulate plant growth and development either by releasing plant growth regulators; solubilizing phosphorus, potassium, zinc, selenium and silicon; biological nitrogen fixation and production of siderophores, ammonia, hydrogen cyanide, hydrolytic enzymes and bioactive compound/secondary metabolites. Biofertilizer developed using mineral solubilizing microbes is an eco-friendly solution to the sustainable food production system in many countries worldwide. The present review deals with the biodiversity of mineral solubilizing microbes, and potential roles in crop improvement and soil well-being for agricultural sustainability.     

Classification of large array surface myoelectric potentials from subjects with and without low back pain.

An algorithm was developed and tested for differentiating between the spatial distribution of large arrays of surface electromyographic (LASE) data from subjects with and without low back pain (LBP). The surface EMG data from 62 channels were collected from the low back of 161 healthy and 44 acute (less than 6-weeks) LBP subjects in three minimum stress postural positions including standing, 20 degrees of trunk flexion (at hip joint) and standing with arms extended forward holding a 1.36kg (3lb) weight in each hand. These data were statistically analyzed and the spatial distribution of the root mean square (RMS) values was used in a multivariate quadratic discriminant model to reclassify the healthy and acute LBP subjects. The most predictive results were obtained from the 'flexion' group of experiments and correctly reclassified 95.5% (42/44) of the acute LBP subjects and 99.4% (160/161) of the healthy subjects. The success rate of this reclassification based on surface distribution of myoelectric potentials was found to be better than the reported patient classifications based on a smaller set of electrode pairs using fewer subjects [Peach JP, McGill SM, Classification of low back pain with use of spectral electromyogram parameters. Spine 23(10):1998;1117-23; Roy SH, De Luca CJ, Emley M, Oddsson LI, Buijs RJ, Levins JA, Newcombe DS, Jabre JF. Classification of back muscle impairment based on the surface electromyographic signal. J Rehabil Res Dev 34(4):1997;405-14 [review]]. The results indicated the potential of the model for clinical patient classification.     

The carboxyl terminus controls ligand-dependent activation of VEGFR-2 and its signaling

Vascular endothelial growth factor receptor-2 (VEGFR-2/FLK-1) is a receptor tyrosine kinase whose activation stimulates angiogenesis. We recently generated a chimeric VEGFR-2 in which the extracellular domain of VEGFR-2 was replaced with the extracellular domain of human colony stimulating factor-1 receptor and expressed in endothelial cells. To study the contribution of the carboxyl terminus to activation of VEGFR-2, we created a panel of truncated receptors in which the carboxyl terminus of VEGFR-2 was progressively deleted. Removal of the entire carboxyl terminus eliminated activation of VEGFR-2, its ability to activate signaling proteins, and its ability to stimulate cell proliferation. The carboxyl terminus-deleted VEGFR-2 exhibited impaired ligand-dependent down-regulation and inhibited the activation of wild-type receptor in a dominant-negative fashion. Furthermore, introducing the carboxyl terminus of another receptor, i.e., VEGFR-1, restored the ligand-dependent activation of the carboxyl terminus-deleted VEGFR-2 and its ability to stimulate cell proliferation. Our findings suggest that the carboxyl terminus of VEGFR-2 plays a critical role in VEGFR-2 activation, its ability to activate signaling proteins, and its ability to induce biological responses. The presence of at least 57 amino acids at the carboxyl terminus of VEGFR-2 are required for VEGFR-2 activation. Thus, we propose that the carboxyl terminus is required for activation of VEGFR-2, and absence of the carboxyl terminus renders VEGFR-2 inactive.     

Observation of an arsenic adduct in an acetyl esterase crystal structure

The crystal structures of an acetyl esterase, HerE, and its complex with an inhibitor dimethylarsinic acid have been determined at 1.30- and 1.45-A resolution, respectively. Although the natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield phenol. Recently, the activity of the enzyme toward heroin has been exploited to develop a heroin biosensor, which affords higher sensitivity than other currently available detection methods. The crystal structure reveals a single domain with the canonical alpha/beta hydrolase fold with an acyl binding pocket that snugly accommodates the acetyl substituent of the substrate and three backbone amides that form a tripartite oxyanion hole. In addition, a covalent adduct was observed between the active site serine and dimethylarsinic acid, which inhibits the enzyme. This crystal structure provides the first example of an As-containing compound in a serine esterase active site and the first example of covalent modification of serine by arsenic. Thus, the HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds.     

Assignment validation software suite for the evaluation and presentation of protein resonance assignment data

We present a set of utilities and graphical user interface (GUI) tools for evaluating the quality of protein resonance assignments. The Assignment Validation Software (AVS) suite, together with new GUI features in the AutoAssign software package, provides a set of reports and graphs for validating protein resonance assignment data before its use in structure analysis and/or submission to the BioMagResBank (BMRB). Input includes a listing of resonance assignments and a summary of sequential connectivity data (i.e. triple resonance, NOE, or other data) used in deriving the assignments. These tools are useful for evaluating the accuracy of protein resonance assignments determined by either automated or manual methods.     

Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.